Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.Fanconi anemia (FA)³ is a genetic disorder characterized by chromosome instability, predisposition to cancer, hypersensitivity to DNA cross-linking agents, developmental abnormalities, and bone marrow failure (1–9). There are at least 13 distinct FA complementation groups, each of which is associated with an identified gene (2, 9, 10). Eight of them are components of the FA core complex (FANC A, B, C, E, F, G, L, and M) that is epistatic to the monoubiquitination of both FANCI and FANCD2, a key event to initiate interstrand cross-link (ICL) repair (2, 9, 11). Downstream of or parallel to the FANCI and FANCD2 monoubiquitination are the proteins involved in double strand break repair and breast cancer susceptibility (i.e. FANCD1/BRCA2, FANCJ/BRIP1, and FANCN/PALB2) (2, 9).FANCI is the most recently identified FA gene (11–13). FANCI protein is believed to form a FANCI-FANCD2 (ID) complex with FANCD2, because they co-immunoprecipitate with each other from cell lysates and their stabilities are interdependent of each other (9, 11, 13). FANCI and FANCD2 are paralogs to each other, since they share sequence homology and co-evolve in the same species (11). Both FANCI and FANCD2 can be phosphorylated by ATR/ATM (ataxia telangiectasia and Rad3-related/ataxia telangiectasia-mutated) kinases under genotoxic stress (11, 14, 15). The phosphorylation of FANCI seems to function as a molecular switch to turn on the FA repair pathway (16). The monoubiquitination of FANCD2 at lysine 561 plays a critical role in cellular resistance to DNA cross-linking agents and is required for FANCD2 to form damage-induced foci with BRCA1, BRCA2, RAD51, FANCJ, FANCN, and γ-H2AX on chromatin during S phase of the cell cycle (17–25). In response to DNA damage or replication stress, FANCI is also monoubiquitinated at lysine 523 and recruited to the DNA repair nuclear foci (11, 13). The monoubiquitination of both FANCI and FANCD2 depends on the FA core complex (11, 13, 26), and the ubiquitination of FANCI relies on the FANCD2 monoubiquitination (2, 11). In an in vitro minimally reconstituted system, FANCI enhances FANCD2 monoubiquitination and increases its specificity toward the in vivo ubiquitination site (27).FANCI is a leucine-rich peptide (14.8% of leucine residues) with limited sequence information to indicate which processes it might be involved in. Besides the monoubiquitination site Lys⁵²³ and the putative nuclear localization signals (Fig. 1A), FANCI contains both ARM (armadillo) repeats and a conserved C-terminal EDGE motif as FANCD2 does (11, 28). The EDGE sequence in FANCD2 is not required for monoubiquitination but is required for mitomycin C (MMC) sensitivity (28). The ARM repeats form α-α superhelix folds and are involved in mediating protein-protein interactions (11, 29). In addition, FANCI, at its N terminus, contains a leucine zipper domain (aa 130–151) that could be involved in mediating protein-protein or protein-DNA interactions (Fig. 1A) (30–33). FANCD2, the paralog of FANCI, was reported to bind to double strand DNA ends and Holliday junctions (34).Open in a separate windowFIGURE 1.Purified human FANCI binds to DNA promiscuously. A, schematic diagram of predicted FANCI motifs and mutagenesis strategy to define the DNA binding domain. The ranges of numbers indicate how FANCI was truncated (e.g. 801–1328 represents FANCI-(801–1328)). NLS, predicted nuclear localization signal (aa 779–795 and 1323–1328); K523, lysine 523, the monoubiquitination site. The leucine zipper (orange bars, aa 130–151), ARM repeats (green bars), and EDGE motif (blue bars) are indicated. Red bars with a slash indicate the point mutations shown on the left. B, SDS-PAGE of the purified proteins stained with Coomassie Brilliant Blue R-250. R1285Q and D1301A are two point mutants of FANCI. All FANCI variants are tagged by hexahistidine. FANCD2 is in its native form. Protein markers in kilodaltons are indicated. C, titration of WT-FANCI for the DNA binding activity. Diagrams of the DNA substrates are shown at the top of each set of reactions. *, ³²P-labeled 5′-end. HJ, Holliday junction. Concentrations of FANCI were 0, 20, 40, 60, and 80 nm (ascending triangles). The substrate concentration was 1 nm. Protein-DNA complex is indicated by an arrow. D, supershift assay. 1 nm of ssDNA was incubated with PBS (lane 1), 80 nm FANCI alone (lane 2), and 80 nm FANCI preincubated with a specific FANCI antibody (lane 3) in the condition described under “Experimental Procedures.”In order to delineate the function of FANCI protein, we purified the recombinant FANCI from the baculovirus expression system. In this study, we report the DNA binding activity of FANCI. Unlike FANCD2, FANCI binds to different DNA structures, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), 5′-tailed, 3′-tailed, splayed arm, 5′-flap, 3′-flap, static fork, and Holliday junction with preference toward branched structures in the presence of FANCD2. Our data suggest that the dynamic DNA binding activity of FANCI and the preferential recognition of branched structures by the ID complex are likely to be the mechanisms to initiate downstream repair events.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.Tissue imaging by mass spectrometry (MS) is proving useful in areas such as detecting tumor margins, determining sites of high drug uptake, and mapping signaling molecules in brain tissue (1–8). Imaging using secondary ion mass spectrometry is well established but is only marginally useful with intact molecular mass measurements from biological tissue (9–11). Matrix-assisted laser desorption/ionization (MALDI)-MS operating under vacuum conditions has been used for tissue imaging with success, especially for abundant components such as membrane lipids, drug metabolites, and proteins (12–14). Spatial resolution of ∼20 μm has been achieved (15), and the MALDI-MS method has been applied in an attempt to shed light on Parkinson disease (16, 17), muscular dystrophy (18), obesity, and cancer (12, 19).Unfortunately, there are disadvantages in using vacuum-based MS for tissue imaging in relation to analysis of unadulterated tissue. Also, the mass spectrometers used in these studies frequently have much lower mass resolution and mass accuracy than are available with atmospheric pressure ionization (API)¹ instruments and are not as widely available. Because the vacuum ionization methods produce singly charged ions, mass-selected fragmentation methods provide only limited information, especially for proteins. In addition, no advanced fragmentation such as electron transfer dissociation (ETD) (20–22) is available for confident protein confirmation or identification. Atmospheric pressure (AP) MALDI can be coupled to high performance mass spectrometers but suffers from sensitivity issues for tissue imaging where high spatial resolution is desired (23). AP MALDI also primarily produces singly charged ions (24, 25). Thus, mass and cross-section analysis of intact proteins has yet to be accomplished using AP MALDI because of intrinsic mass range limitations of API instruments, which frequently have a mass-to-charge (m/z) limit of <4000. Thus, new improved methods of mass-specific tissue imaging, especially at AP, are needed.The potential of laserspray ionization (LSI) (Scheme 1) (26–33) for protein tissue analysis is reported here. LSI has advantages relative to other MS-based methods, including speed of analysis, laser ablation of small volumes, more relevant AP conditions, extended mass range and improved fragmentation through multiple charging, and the ability to obtain cross-section data for proteins on appropriate instrumentation. The applicability of LSI for high mass compounds on high performance API mass spectrometers (Orbitrap Exactive and SYNAPT G2) has been demonstrated producing ESI-like multiply protonated ions (26–28). The first experiments showing sequence analysis by ETD using the LSI method were successfully carried out on a Thermo Fisher Scientific (San Jose, CA) LTQ-ETD mass spectrometer (26). Nearly complete sequence coverage was obtained for ubiquitin, an important regulatory protein. Applying ETD fragmentation to LSI-MS analyses potentially provides a new method for studying biological processes, including the mapping of phosphorylation, glycosylation, and ubiquitination sites from intact proteins and directly from tissue.Open in a separate windowScheme 1.Overview of LSI-MS operated in transmission geometry.Furthermore, unlike ESI and related ESI-based methods such as desorption-ESI (34), the LSI method has been shown to allow analysis of lipids in tissue from ablated areas <80 μm (30). In comparison with literature reports for AP MALDI at the same stage of development (35), LSI is more than an order of magnitude more sensitive and is capable of analyzing proteins on high resolution mass spectrometers as was demonstrated by obtaining full-acquisition mass spectra at 100,000 mass resolution (FWHH, m/z 200) after application of only 20 fmol of bovine pancreas insulin in the matrix 2,5-dihydroxyacetophenone (2,5-DHAP) onto a glass microscope slide (33). The analysis speed of LSI was demonstrated by obtaining mass spectra of five samples in 8 s (32). Here, we show the utility of LSI for intact peptide and protein analyses directly from mouse brain tissue. The ability to obtain a protein mass spectrum directly from mouse brain tissue in a single laser shot at 100,000 mass resolution and with ETD fragmentation is demonstrated.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.     

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu²⁺, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1)³ cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1–10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11–16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These “as-isolated” SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between Cys-57 and Cys-146 (20). The significance of these results is that even WT-like mutants, which exhibit a nearly normal backbone structure (21–23), may be vulnerable to destabilizing influences in vivo. Our group and others subsequently showed that the mutant SOD1 proteins share a susceptibility to increased hydrophobicity under conditions that reduce disulfide bonds and/or chelate metal ions (5) and that similar hydrophobic species exist in tissue lysates from mutant SOD1 transgenic mice (4–6). One consequence of such hydrophobic exposure could be the facilitation of abnormal interactions between the mutant enzymes and other cellular constituents (e.g. chaperones, mitochondrial components, or other targets), which might influence pathways leading to motor neuron death (15, 16, 24–27).Accumulating evidence suggests that metal deficiency of SOD1 is an important factor that can influence SOD1 aggregation or neurotoxicity (4, 28–33), but the metal-deficient states of SOD1 that are most relevant to ALS remain unclear. Zinc-deficient, copper-replete SOD1 species, which can be produced in vitro by adding copper to SOD1 that has been stripped of its metal ions at acidic pH, were shown to be toxic to motor neurons in culture (28). However, it has not been shown that zinc-deficient, copper-replete SOD1 is produced in vivo as a consequence of ALS mutations, and loading of copper into SOD1 by the copper chaperone for SOD1 (CCS) is not required for toxicity (34, 35). Furthermore, the MBR mutants have a disrupted copper site and have been found to be severely deficient in both zinc and copper (17, 30), yet expression of these SOD1s still produces motor neuron disease (1, 2, 30, 34, 36, 37).When recombinant human SOD1 was overexpressed in insect cells, we instead observed zinc-replete but copper-deficient species for most WT-like mutants, probably because the capacity of the copper-loading mechanism was exceeded (17). These preparations indicate that zinc can be efficiently incorporated into many WT-like mutants in vivo, and much of it is retained after purification. Furthermore, these copper-deficient biologically metallated proteins may be useful reagents to assess the influence of copper binding upon other properties of SOD1 mutants that may be relevant to their neurotoxicity.We previously observed that reduction of the Cys-57—Cys-146 disulfide bond facilitates the ability of metal chelators to alter the electrophoretic mobility and to increase the hydrophobicity of SOD1 mutants (5). This is consistent with the known properties of this linkage to stabilize the dimeric interface, to orient Arg-143 via a hydrogen bond from the carbonyl oxygen of Cys-57 to Arg-143-NH₂, and to prevent metal ion loss (38–40). However, it remains unclear whether the Cys-57—Cys-146 bond is required to prevent abnormal SOD1 hydrophobic exposure or whether the aberrant conformational change primarily results from metal ion loss. Ablation of the disulfide bond by the experimental (non-ALS) mutants C57S and C146S provides useful reagents to test the relative influence of the disulfide bond and copper binding upon SOD1 properties.In this study we sought to correlate the consequences of copper deficiency, copper and zinc deficiency, and disulfide reduction upon the hydrodynamic behavior and surface hydrophobicity of WT and representative mutant SOD1 enzymes (Fig. 1A). We quantitated the metal contents of as-isolated SOD1 proteins, detected changes in conformation or metal occupancy using native PAGE to assess their electrophoretic mobility, a measure of global conformational change, and correlated these changes to hydrophobic exposure using 1-anilinonaphthalene-8-sulfonic acid (ANS), which is very sensitive to local conformational changes. ANS is a small amphipathic dye (Fig. 1B) that has been used as a sensitive probe to detect hydrophobic pockets on protein surfaces (41–44). Free ANS exhibits only weak fluorescence that is maximal near 520 nm, but when ANS binds to a hydrophobic site in a partially or fully folded protein, the fluorescence peak increases in amplitude and shifts to a shorter wavelength (42). ANS also has an anionic sulfonate group that can interact with cationic groups (e.g. Arg or Lys residues) through ion-pair formation which may be further strengthened by hydrophobic interactions (43–46).Open in a separate windowFIGURE 1.A, WT SOD1 structure showing the position of the C57-C146 intrasubunit disulfide bond (S–S, yellow), bound copper and zinc ions, and ALS mutant residues. The residues altered in A4V, G85R, G93A, D124V, and S134N SOD1s are indicated as green spheres. The backbone of the β-barrel core and the loops is shown in a rainbow color, from blue at the amino terminus to red at the carboxyl terminus. The figure was generated using PyMOL (84) and PDB entry 1HL5 (22). B, chemical structure of ANS fluorophore.To evaluate further the importance of metal ion binding, we measured spectral changes related to the binding of cobalt and copper to the same SOD1 proteins. We observed that as-isolated WT-like mutants containing zinc could interact with copper ions to produce an electrophoretic mobility and decreased hydrophobicity resembling that of the fully metalated holo-WT SOD1. In contrast, we saw no evidence for copper binding to MBR mutants in a manner that alters their hydrodynamic properties or their hydrophobicity. Our data suggest that binding of both copper and zinc are important determinants of SOD1 conformation and that perturbation of such binding may be relevant to the ALS disease process.     

PR55��, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated ��-Catenin Dephosphorylation

A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (1–3). Our previous work found that β-catenin is sequentially phosphorylated by CKIα⁴ and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.